X-chromosome inactivation results in the same dosage of gene expression between males and females of mammals. However, not all X-linked genes are subject to X inactivation as recently shown by the finding of several genes that escape X inactivation in human. It is not known whether genes that escape X inactivation in adult do so from the onset of inactivation in embryogenesis. Of special interest is the XIST gene that is expressed only from the inactive X chromosome and may play a role in the onset of X inactivation. In this proposal, we outline experiments to examine in vivo the X-inactivation status of genes in adult and embryo mice. We plan to isolate new mouse genes that escape X inactivation from a human x mouse hybrid cell line that retains only the inactive mouse X chromosome under selective pressure for the neomycin-resistance gene inserted in that chromosome. Mouse-specific transcripts corresponding to genes that escape X inactivation will be isolated from a cDNA library constructed from the hybrid cell line. We will then map, by in situ hybridization to mouse chromosomes, the new genes isolated from the hybrid cell line and existing X-linked genes known to escape X inactivation in human. In addition to locating the genes in mouse, this analysis may reveal the presence of Y homologs. We will determine the inactivation status of the genes in adult mice in vivo, by exploiting a mouse X-autosome translocation where the normal X chromosome is inactive in all cells and the genetic variation between mouse species to evaluate allelic expression at a given locus by a reverse transcriptase polymerase chain reaction assay. We will extend these studies to the mouse embryo by taking advantage of the preferential paternal X- chromosome inactivation in extraembryonic membranes. We will follow the expression of Xist in embryos to see whether the onset of its expression correlates with that X inactivation. The expression of a transgene previously shown to escape X inactivation in adult mouse will be followed during embryogenesis to determine whether there is reactivation of the transgene or whether it escapes inactivation from the onset. Finally, we will look for regions of early replication, in the otherwise late-replicating inactive mouse X chromosome, that may delineate chromosomal regions that contain genes that escape X inactivation.